IMAC purified STNhaA (7 µg) was incubated on ice for 45 min in presence of 1x sample buffer (Invitrogen, BN2003) and 3x cathode buffer (Invitrogen, BN2002). After centrifugation at 10,000×g for 5 min at 4°C, supernatant was loaded on a 4–16% BisTris NativePage gel (Invitrogen) and separated in Dark Blue Buffer (Invitrogen) for 2 h at 150 V.
Bn2003
Manufactured by Thermo Fisher Scientific
The BN2003 is a laboratory device designed for the measurement and analysis of molecular properties. It provides precise quantification and characterization of biopolymers such as proteins, nucleic acids, and other macromolecules. The core function of the BN2003 is to perform analytical tasks through advanced spectroscopic techniques.
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Lab products found in correlation
G 250 sample additive, by Thermo Fisher Scientific (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Bis tris native page, by Thermo Fisher Scientific (1 mentions)
Ab14715, by Abcam (1 mentions)
Ab14744, by Abcam (1 mentions)
Ab15191, by Abcam (1 mentions)
D4641, by Merck Group (1 mentions)
Bn1004box, by Thermo Fisher Scientific (1 mentions)
Nupage transfer buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bn1002box, by Thermo Fisher Scientific (1 mentions)
Coomassie g 250, by Thermo Fisher Scientific (1 mentions)
Lc0725, by Thermo Fisher Scientific (1 mentions)
Bn2007, by Thermo Fisher Scientific (1 mentions)
4 protocols using bn2003
1
Native PAGE Analysis of STNhaA
2
Mitochondrial Protein Complex Analysis
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Mitochondria were solubilized in a cold 1 × sample buffer (BN2003, Invitrogen) containing 2% n-dodecyl-β-D-maltoside (DDM, D4641, Sigma) and 5% G-250 Sample Additive (BN2004, Invitrogen). The lysates were centrifuged at 20,000 × g for 30 min at 4°C after incubation on ice for 15 min. The protein concentration was determined by the BCA protein assay (23227, Thermo Scientific), with 5 μg of protein loaded in each lane of the gel. CIV was analyzed by blue native-polyacrylamide gel electrophoresis (BN-PAGE) using a linear 4–16% gradient gel (BN1004BOX, Invitrogen) and transferred to polyvinylidene fluoride membranes by western blotting at a constant voltage of 25 V for 1 h. The membranes were then incubated in 20 ml of 8% acetic acid for 15 min to fix the proteins and blocked with 5% non-fat milk. After blocking, the membranes were incubated with primary antibodies against COX2 (1:1,000, ab15191, Abcam) and COX4 (1:1,000, ab14744, Abcam), followed by incubation with secondary antibodies from the same species. Complex II (CII) (1:1,000, ab14715, Abcam) was used as a loading control.
3
Native PAGE Analysis of IP3R Complexes
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HEK-3KO and stable cell lines expressing WT or mutant IP3R1/3 constructs were lysed in CHAPS lysis buffer supplemented with protease inhibitors (Roche). After 15 min on ice, cell lysates were cleared by centrifugation at 13,000 rpm for 10 min at 4 °C. Next, 15 µg cleared lysates were mixed with 4X sample buffer (BN2003, Invitrogen) and 5% G-250 sample additive (BN2004, Invitrogen). Samples were separated at 4 °C on a 3 to 12% Native PAGE Novex (BN2011BX10, Invitrogen) at 150 V for 1.5 h using dark cathode buffer and then at 250 V for 1 h using light cathode buffer (BN2007, Invitrogen) in the inner chamber with anode buffer in the outer chamber. Separated proteins were transferred onto polyvinylidene difluoride membrane using NuPAGE Transfer buffer (NP0006-1, Invitrogen) overnight at 4 °C. Appropriate primary and secondary antibodies were used to detect IP3R1/3 complexes, as indicated for western blotting. Molecular weight markers are based on the mobility of unstained protein standard (LC075, Invitrogen).
4
Purification and Native-PAGE of Spartin Proteins
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Spartin proteins including HsSpartin, CeSpartin and CtSpartinL were expressed in Expi293 cells, and purified using buffer A (50 mM HEPES, 500 mM Nacl, pH8.0, 1 mM TCEP, 10% glycerol) using anti-FLAG resin. Proteins were quantified using BSA standards in SDS-PAGE. Proteins (1–2 μg) together with BSA (1 μg) were loaded onto native PAGE 4%−16% Bis-Tris gel (BN1002BOX, invitrogen) for electrophoresis at 4C using NativePAGE running buffer and NativePAGE Cathode buffer (contains 0.02% Coomassie G-250, BN2007, invitrogen). NativeMark unstained protein standards (LC0725, invitrogen) and NativePAGE sample buffer (BN2003, invitrogen) were used. Protein gels were fixed and destained according to manufacturer direction. Distinct from native gels in the lipid-co-migration assay (above), where samples migrate according to their charge/mass ratio, here the Coomassie dye in the cathode buffer coats protein sample, which consequently migrates according to MW (28 (link)).
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